CN103444523B - Method for quickly introducing embryonic callus through anther to regenerate plant - Google Patents

Method for quickly introducing embryonic callus through anther to regenerate plant Download PDF

Info

Publication number
CN103444523B
CN103444523B CN201310026841.5A CN201310026841A CN103444523B CN 103444523 B CN103444523 B CN 103444523B CN 201310026841 A CN201310026841 A CN 201310026841A CN 103444523 B CN103444523 B CN 103444523B
Authority
CN
China
Prior art keywords
callus
grape
culture
anther
root
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CN201310026841.5A
Other languages
Chinese (zh)
Other versions
CN103444523A (en
Inventor
赵凤霞
高相彬
王正平
李耀宇
宋鹏飞
李海峰
宋学立
孟智勇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tobacco Research Institute Henan Academy Of Agricultural Sciences
Original Assignee
Tobacco Research Institute Henan Academy Of Agricultural Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tobacco Research Institute Henan Academy Of Agricultural Sciences filed Critical Tobacco Research Institute Henan Academy Of Agricultural Sciences
Priority to CN201310026841.5A priority Critical patent/CN103444523B/en
Publication of CN103444523A publication Critical patent/CN103444523A/en
Application granted granted Critical
Publication of CN103444523B publication Critical patent/CN103444523B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Landscapes

  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The invention provides a method for quickly introducing embryonic callus through anther to regenerate a plant, and belongs to the field of agricultural science. The method comprises the following steps: getting wine grape Riesling anther as a material; inducing the embryonic callus through the anther; performing differentiation culture on the callus; performing rooting culture on the differentiated seedlings; and acclimatizing and transplanting to accomplish the grape plant regeneration. The culture medium and the cultivation method have good effect, and have the advantages that the inductivity of the embryonic callus is high, the quality is high, and the cultivation cycle is relatively short; simultaneously, subculturing can be performed for a long term, and a constant loose embryonic callus state can be kept, therefore, sufficient materials are prepared for the study on production of secondary metabolite for grape cell culture, and the study on biotechnology. The culture medium and the anther cultivation method applied to grape genetic breeding can shorten the breeding cycle and provide an important technology platform for improving the grape varieties.

Description

A kind of method of flower pesticide rapid induction embryo callus regeneration plant
Technical field
The invention belongs to field of tissue culture, be specifically related to a kind of method of cultivating induction grape flower pesticide formation embryo callus by tissue.
Background technology
Set up the grape regenerating system of stablize high frequency for optimizing transgenosis condition, raising transgene efficiency, carries out new grape variety seed selection and genetic improvement significant.Carry out gene genetic Study on Transformation taking callus as acceptor material and there are a lot of advantages.The first, the cell having broken up is all returned to the meristematic cell level of dedifferentiation, is easy to accept foreign gene, and transformation efficiency is higher; The second, expand numerous amount large, the transformed calli of acquisition expands numerous cultivation by subculture, can differentiate more transformed plant; The 3rd, Various Tissues, organ all can evoked callus, and explant examination material is extensive.The acquisition of grape regenerating system is at present mainly by two approach: neomorph approach and somatic embryo Regeneration Ways.Neomorph approach is direct indefinite bud, the adventive root of forming on explant, the whole plant of regenerating, or explant process dedifferentiation induction generation callus, thus callus breaks up acquisition regeneration plant on differential medium.This approach regenerative process is simple, and the cycle is short, but easily produces chimera.Somatic embryo Regeneration Ways is directly to differentiate somatic embryo or plant tissue is first induced generation callus by plant tissue, and callus is through induction organizator blast.Producing somatic embryo by organ induced embryonic callus such as flower pesticide, filigree, ovules is best, the most widely used induction mode of effect in the research of grape regenerating system.Induction Process exists embryonic callus induction efficiency low, is accompanied by the appearance of non-embryonic callus tissue, and cultivation period is long etc., has limited the application of grape anther culture in actual breeding.Therefore the inductivity that improves grape embryo callus is a key issue in the research of grape anther culture technique.
Summary of the invention
The object of the invention is to overcome the low deficiency of existing grape flower pesticide induced embryonic callus inductivity, provide a kind of efficient induction grape flower pesticide to form culture medium prescription and the abductive approach of embryo callus and then regeneration grapevine seedling, improving the inductivity of embryo callus by the method, is to lay the foundation for setting up efficient grape anther culture plant regeneration.
The present invention is achieved through the following technical solutions:
The method of grape flower pesticide rapid induction embryo callus regeneration plant: utilizing wine grape Riesling flower pesticide is material, carries out the steps such as flower pesticide induced embryonic callus, Calli Differentiation cultivation, the cultivation of differentiation seedling rooting, acclimatization and transplants and completes grapevine seedling regeneration.According to the method, it is characterized in that grape is contained to spend front 12-14 days, pollen tetrad, to monokaryotic stage, is won the full bud of flower pesticide in the fine morning, and 4 DEG C of dark preservations 3 days can improve the induction efficiency of embryo callus; From sterile bud, take out flower pesticide and be inoculated in callus inducing medium under aseptic condition, callus inducing medium formula is: MS macroelement, MS trace element, MS molysite, 0.5mg L -1thiamine hydrochloride, 0.2mg L -1puridoxine hydrochloride, 0.2mg L -1nicotinic acid, 150mgL -1inositol, 2.0mg L- 1p-CPA, 0.20mg L -1tDZ, 5mg L -1agNO 3, 10mg L -1gentamicin, 100mg L -1cysteine, 50mg L -1caseinhydrolysate, 3% glucose and 0.28% de-acetyl gellan gum, pH 6.2.Embryo callus is inoculated into organize on differential medium and carries out induction, organize differential medium to be: MS macroelement, MS trace element, MS molysite, 0.5mg L -1thiamine hydrochloride, 0.2mg L -1puridoxine hydrochloride, 0.5mg L -1nicotinic acid, 150mg L -1inositol, 1.0mg L -1p-CPA, 0.20mg L -1tDZ, 0.5mg L -1nOA, 0.01mg L -1gA 3, 50mg L -1caseinhydrolysate, 2.0mg L -1glycine, 10g L -1coconut palm breast, 5% sucrose and 0.28% de-acetyl gellan gum, pH 6.2.Condition of culture is 25 DEG C of temperature, humidity 70%, illumination 1000-1500lux, light application time 12 hours/day.Differentiation seedling is proceeded to root media root induction, and culture of rootage based component is 1/2MS macroelement, MS trace element, MS molysite, 0.1mg L -1thiamine hydrochloride, 0.8mg L -1puridoxine hydrochloride, 0.5mgL -1nicotinic acid, 100mg L -1inositol, 0.5mg L -1α-naphthaleneacidsodium, 0.5mg L -1nOA, 50mg L -1caseinhydrolysate, 2.0mg L -1glycine, 3% glucose and 0.28% de-acetyl gellan gum, pH 6.2.Condition of culture is for first cultivating under light one week, more secretly cultivates one week, then under light, is cultured to and normally takes root, 25 DEG C of temperature, humidity 70%, illumination 1500-2000lux, light application time 12 hours/day; When seedling base portion grows 3-4 bar root, when the long 3cm of root, open bottle cap, 25 DEG C of environmental temperatures, humidity 85%, intensity of illumination 2000lux, hardening 5 days; Then taking-up group training seedling, running water is cleaned root medium, is transplanted in sterilized pearlite interstitial substance, after 1/8MS nutrient solution irrigates, water weekly culture fluid one time, add a cover with the plastic sack of the upper end clip of nutritive cube formed objects and keep appropriateness, 25 DEG C of indoor cultivations 1 month, after be transplanted to land for growing field crops.
Further, the method is carried out according to following steps:
(1) flower pesticide collection and disinfecting: grape is contained and spent front 12-14 days, and pollen tetrad, to monokaryotic stage, is won the full bud of flower pesticide in the fine morning, 4 DEG C of dark preservations 3 days, then carry out surface sterilization to bud.
(2) anther callus is cultivated: from sterile bud, take out flower pesticide and be inoculated in callus inducing medium under aseptic condition.Described callus inducing medium formula is: MS macroelement, MS trace element, MS molysite, 0.5mg L -1thiamine hydrochloride, 0.2mg L -1puridoxine hydrochloride, 0.2mg L -1nicotinic acid, 150mg L -1inositol, 2.0mg L -1p-CPA, 0.20mg L -1tDZ, 5mg L -1agNO 3, 10mgL -1gentamicin, 100mg L -1cysteine, 50mg L -1caseinhydrolysate, 3% glucose and 0.28% de-acetyl gellan gum, pH 6.2.
(3) Calli Differentiation is cultivated: embryo callus is inoculated into organize and on differential medium, carries out induction.The described differential medium formula of organizing is: MS macroelement, MS trace, MS molysite, 0.5mgL -1thiamine hydrochloride, 0.2mgL -1puridoxine hydrochloride, 0.5mgL -1nicotinic acid, 150mg L -1inositol, 1.0mg L -1p-CPA, 0.20mg L -1tDZ, 0.5mg L -1nOA, 0.01mg L -1gA 3, 50mgL -1caseinhydrolysate, 2.0mg L -1glycine, 10g L -1coconut palm breast, 5% sucrose and 0.28% de-acetyl gellan gum, pH 6.2.Condition of culture is 25 DEG C of temperature, humidity 70%, illumination 1000-1500lux, light application time 12 hours/day.
(4) differentiation seedling rooting is cultivated: differentiation seedling is proceeded to root media root induction, and described culture of rootage based component is 1/2MS macroelement, MS trace element, MS molysite, 0.1mg L -1thiamine hydrochloride, 0.8mgL -1puridoxine hydrochloride, 0.5mgL -1nicotinic acid, 100mgL -1inositol, 0.5mg L -1α-naphthaleneacidsodium, 0.5mg L -1nOA, 50mg L -1caseinhydrolysate, 2.0mg L -1glycine, 3% glucose and 0.28% de-acetyl gellan gum, pH 6.2.Condition of culture is for first cultivating under light one week, more secretly cultivates one week, then under light, is cultured to and normally takes root, 25 DEG C of temperature, humidity 70%, illumination 1500-2000lux, light application time 12 hours/day.
(5) hardening, heel in, transplant: when seedling base portion grows 3-4 bar root, when the long 3cm of root, open bottle cap, 25 DEG C of environmental temperatures, humidity 85%, intensity of illumination 2000lux, hardening 5 days; Then taking-up group training seedling, running water is cleaned root medium, is transplanted in sterilized pearlite interstitial substance, after 1/8MS nutrient solution irrigates, water weekly culture fluid one time, add a cover with the plastic sack of the upper end clip of nutritive cube formed objects and keep appropriateness, 25 DEG C of indoor cultivations 1 month, after be transplanted to land for growing field crops.
Effect of the present invention:
Compared with the prior art, the present invention adopts and contains the grape bud of spending front 12-14 days, before inoculation, bud is carried out to 4 DEG C of dark preservations 3 days, is conducive to the raising of embryonic callus induction efficiency.Callus inducing medium, compared with common MS medium, contains higher thiamine hydrochloride and inositol, has improved the induction efficiency of embryo callus; The growth hormone of higher concentration is conducive to evoked callus and forms; Gentamicin has alleviated the pollution that manual operation causes to a certain extent; AgNO 3alleviate callus browning necrosis with cysteine; Organize differential medium compared with common MS medium, the thiamine hydrochloride of higher concentration and inositol and additional coconut palm breast are conducive to the differentiation of callus and the formation of embryoid, the sucrose of high concentration (5%) is conducive to the growth of embryoid, the gibberellin GA of trace 3be conducive to the growth of embryoid; The puridoxine hydrochloride (8%) that root media contains higher concentration compared with common MS medium, is conducive to the growth of root system; The present invention adopts 0.28% de-acetyl gellan gum as curing agent, and compared with agar, de-acetyl gellan gum transparency is high, good permeability, solidifying low with point, few containing impurity components such as Ca, Mg, be conducive to contained nutriment in medium and be absorbed by plants, there is higher value.
Utilize medium of the present invention to carry out the anther cultural method of grape and have advantages of that frequency of embryonic callus induction is high, cultivation cycle is short, the inductivity of embryo callus is between 40%-60%, and cultivation cycle is about 7 months.Flower pesticide is drawn materials conveniently, simple in structure, and genotype is abundant, and method is easy, easy operating and application.
Brief description of the drawings:
Fig. 1: flower pesticide evoked callus figure;
Fig. 2: Calli Differentiation figure;
Fig. 3: culture of rootage seedling.
Embodiment:
1, flower pesticide collection and disinfecting:
(1) flower pesticide collection: grape is contained and spent front 12-14 days, and pollen tetrad, to monokaryotic stage, is won the full bud of flower pesticide in the fine morning, 4 DEG C of dark preservations 3 days.
(2) flower pesticide is disinfected: bud is carried out to surface sterilization, i.e. first immersion treatment 1 minute in the alcohol of 75% (v/v), use again aseptic water washing 3 times, then use the clorox of 2% (v/v) to disinfect 8 minutes, finally use aseptic water washing 5 times.
(3) inoculation: get sterile bud, in super-clean bench under aseptic condition, remove petal by microscope with dissecting needle, clamp filigree with tweezers, take out flower pesticide Direct Uniform and be inoculated in diameter and be that in the culture dish that 9cm contains callus inducing medium, (callus inducing medium is: MS macroelement, MS trace element, MS molysite, 0.5mg L -1thiamine hydrochloride, 0.2mg L -1puridoxine hydrochloride, 0.2mg L -1nicotinic acid, 150mg L -1inositol, 2.0mg L -1p-CPA, 0.20mg L -1tDZ, 5mg L -1agNO 3, 10mg L -1gentamicin, 100mg L -1cysteine, 50mg L -1caseinhydrolysate, 3% glucose and 0.28% de-acetyl gellan gum, pH 6.2).
2, anther callus is cultivated: culture dish is inverted, and 25 DEG C of dark culturing, and a situation arises to observe flower pesticide situation of change and callus, can observe callus appearance after 20 days.Induction Process produces embryo and non-embryo two class callus, and embryo callus mostly is yellow particle shape, and quality is tight, and non-embryonic callus is organized as translucent pasty state, short texture, and quality is soft amorphous.Under microscope, distinguish qualification embryo callus and non-embryonic callus tissue, by the embryo callus subculture amplification of identifying, every 4 weeks subcultures once.
3, Calli Differentiation is cultivated: embryo callus is inoculated into organize and on differential medium, breaks up cultivation (tissue differential medium is: MS macroelement, MS trace element, MS molysite, 0.5mg L -1thiamine hydrochloride, 0.2mg L -1puridoxine hydrochloride, 0.5mg L -1nicotinic acid, 150mg L -1inositol, 1.0mg L -1p-CPA, 0.20mg L -1tDZ, 0.5mg L -1nOA, 0.01mg L -1gA 3, 50mg L -1caseinhydrolysate, 2.0mg L -1glycine, 10g L -1coconut palm breast, 5% sucrose and 0.28% de-acetyl gellan gum, pH 6.2).Produce loose, scleroid cells,primordial group after 2 weeks, these cell masses are easy to depart from maternal tissue, develop into gradually spherical, heart-shaped, cotyledon shape embryoid, break up seedling.Condition of culture is 25 DEG C of temperature, humidity 70%, illumination 1000-1500lux, light application time 12 hours/day.
4, differentiation seedling rooting is cultivated:
Differentiation seedling is proceeded to root media root induction, and (culture of rootage based component is 1/2MS macroelement, MS trace element, MS molysite, 0.1mg L -1thiamine hydrochloride, 0.8mg L -1puridoxine hydrochloride, 0.5mg L -1nicotinic acid, 100mg L -1inositol, 0.5mg L -1α-naphthaleneacidsodium, 0.5mg L -1nOA, 50mg L -1caseinhydrolysate, 2.0mg L -1glycine, 3% glucose and 0.28% de-acetyl gellan gum, pH 6.2).Condition of culture is under first light, to cultivate one week, more secretly cultivates one week, then is cultured to normally and takes root under light, 25 DEG C of temperature, humidity 70%, illumination 1500-2000lux, light application time 12 hours/day.
5, hardening, heel in, transplant:
When seedling base portion grows 3-4 bar root, when the long 3cm of root, open bottle cap, 25 DEG C of environmental temperatures, humidity 85%, intensity of illumination 2000lux, hardening 5 days; Then taking-up group training seedling, running water is cleaned root medium, be transplanted in sterilized pearlite interstitial substance, after 1/8MS nutrient solution irrigates, water weekly culture fluid one time, add a cover with the plastic sack of the upper end clip of nutritive cube formed objects and keep appropriateness, 25 DEG C of indoor cultivations 1 month, after be transplanted to land for growing field crops.
Utilize said method, the present invention is by Riesling grape flower pesticide induced embryonic callus and obtain regeneration plant, and embryonic callus induction efficiency is higher, and between 40%-60%, cultivation cycle is short, about 7 months.

Claims (1)

1. the method for a grape flower pesticide rapid induction embryo callus regeneration plant, it is characterized in that: utilizing wine grape Riesling flower pesticide is material, carry out that flower pesticide induced embryonic callus, Calli Differentiation are cultivated, differentiation seedling rooting is cultivated, acclimatization and transplants step completes grapevine seedling regeneration, it is characterized in that, described method is carried out according to following steps:
(1) flower pesticide collection and disinfecting: grape is contained and spent front 12-14 days, and pollen tetrad, to monokaryotic stage, is won the full bud of flower pesticide in the fine morning, 4 DEG C of dark preservations 3 days, then carry out surface sterilization to bud,
(2) anther callus is cultivated: from sterile bud, take out flower pesticide and be inoculated in callus inducing medium under aseptic condition, described callus inducing medium formula is: MS macroelement, MS trace element, MS molysite, 0.5mg/L thiamine hydrochloride, 0.2mg/L puridoxine hydrochloride, 0.2mg/L nicotinic acid, 150mg/L inositol, 2.0mg/L P-CPA, 0.20mg/L TDZ, 5mg/LAgNO 3, 10mg/L gentamicin, 100mg/L cysteine, 50mg/L caseinhydrolysate, 3% glucose and 0.28% de-acetyl gellan gum, pH 6.2,
(3) Calli Differentiation is cultivated: embryo callus is inoculated into organize on differential medium and carries out induction, the described differential medium formula of organizing is: MS macroelement, MS trace, MS molysite, 0.5mg/L thiamine hydrochloride, 0.2mg/L puridoxine hydrochloride, 0.5mg/L nicotinic acid, 150mg/L inositol, 1.0mg/L P-CPA, 0.20mg/L TDZ, 0.5mg/LNOA, 0.01mg/L GA 3, 50mg/L caseinhydrolysate, 2.0mg/L glycine, 10g/L coconut palm breast, 5% sucrose and 0.28% de-acetyl gellan gum, pH 6.2, condition of culture is 25 DEG C of temperature, humidity 70%, illumination 1000-1500lux, light application time 12 hours/day,
(4) differentiation seedling rooting is cultivated: differentiation seedling is proceeded to root media root induction, described culture of rootage based component is 1/2MS macroelement, MS trace element, MS molysite, 0.1mg/L thiamine hydrochloride, 0.8mg/L puridoxine hydrochloride, 0.5mg/L nicotinic acid, 100mg/L inositol, 0.5mg/L α-naphthaleneacidsodium, 0.5mg/LNOA, 50mg/L caseinhydrolysate, 2.0mg/L glycine, 3% glucose and 0.28% de-acetyl gellan gum, pH 6.2, condition of culture for first cultivating one week under light, secretly cultivate again one week, under light, be cultured to normally and take root again, 25 DEG C of temperature, humidity 70%, illumination 1500-2000lux, light application time 12 hours/day,
(5) hardening, heel in, transplant: when seedling base portion grows 3-4 bar root, when the long 3cm of root, open bottle cap, 25 DEG C of environmental temperatures, humidity 85%, intensity of illumination 2000lux, hardening 5 days; Then taking-up group training seedling, running water is cleaned root medium, is transplanted in sterilized pearlite interstitial substance, after 1/8MS nutrient solution irrigates, water weekly culture fluid one time, add a cover with the plastic sack of the upper end clip of nutritive cube formed objects and keep humidity, 25 DEG C of indoor cultivations 1 month, after be transplanted to land for growing field crops.
CN201310026841.5A 2013-01-08 2013-01-08 Method for quickly introducing embryonic callus through anther to regenerate plant Expired - Fee Related CN103444523B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201310026841.5A CN103444523B (en) 2013-01-08 2013-01-08 Method for quickly introducing embryonic callus through anther to regenerate plant

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201310026841.5A CN103444523B (en) 2013-01-08 2013-01-08 Method for quickly introducing embryonic callus through anther to regenerate plant

Publications (2)

Publication Number Publication Date
CN103444523A CN103444523A (en) 2013-12-18
CN103444523B true CN103444523B (en) 2014-11-12

Family

ID=49727708

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201310026841.5A Expired - Fee Related CN103444523B (en) 2013-01-08 2013-01-08 Method for quickly introducing embryonic callus through anther to regenerate plant

Country Status (1)

Country Link
CN (1) CN103444523B (en)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104686361B (en) * 2015-03-20 2017-01-04 浙江万里学院 The induction of a kind of Fructus Vitis viniferae embryo callus and cultural method
CN111149674A (en) * 2020-02-28 2020-05-15 科稷达隆生物技术有限公司 Culture method for improving transplanting survival rate of plant tissue culture seedlings
CN111758575A (en) * 2020-08-01 2020-10-13 江苏高航农业科技有限公司 Grape anther embryonic callus differentiation culture medium
CN111758574A (en) * 2020-08-01 2020-10-13 江苏高航农业科技有限公司 Grape anther induction culture medium for improving embryogenic callus induction rate
CN115316271A (en) * 2022-07-26 2022-11-11 江苏省农业科学院宿迁农科所 Tissue culture method of muscadine
CN115211370B (en) * 2022-08-18 2023-09-01 西北农林科技大学 Cabernet sauvignon flower organ callus induction medium and culture method
CN115843690B (en) * 2022-12-15 2023-08-08 河北省农林科学院棉花研究所（河北省农林科学院特种经济作物研究所） Regeneration method using honeysuckle anther as explant

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2154626T3 (en) * 1998-05-15 2008-04-16 University Of Florida REGENERATION SYSTEM FOR LIVES AND ITS USES.

Also Published As

Publication number Publication date
CN103444523A (en) 2013-12-18

Similar Documents

Publication Publication Date Title
CN103444523B (en) Method for quickly introducing embryonic callus through anther to regenerate plant
CN101647393B (en) Fast tissue culture reproducing method of actinidia eriantha
CN101933456A (en) Method for quickly breeding seedlings of dendrobium officinale capsule
CN106576910B (en) Culture method of in-vitro sterile host plant-mycorrhizal edible fungus symbiotic seedling
CN105075863B (en) A kind of Paeonia papaveracea rapid propagation method
CN103931492A (en) Tissue-culture rapid seedling growing method for apple rootstock M9
CN102119655A (en) Natural light rapid breeding method for dendrobium officinale
CN110432149B (en) Culture medium and culture method for inducing callus of flax anther and ovary
CN101548647B (en) Tree peony callus induction method
CN104885773A (en) Method for rapidly cultivating early shaping tissue culture commodity seedlings of blueberries
CN103461135A (en) Method for propagating hemerocallis hybridus
CN112189566B (en) Rapid breeding method of cherry seedlings for stocks
CN102550405A (en) Breeding method of poplar haploid
CN103444522B (en) Preparation method for rooting of test-tube plantlet of ample flow pear
CN102805035A (en) Common head cabbage tissue culture method
CN106665357A (en) Method for establishing lycoris regeneration system
CN103155862B (en) Sinocalamus latiflorus flower pesticide inductor embryo the method obtaining regeneration plant
CN106106147B (en) A kind of renovation process of lotus unmature subleaf somatic embryo development ways
CN101124892A (en) Cymbidium edaphic orchids seed aseptic seeding growing seedlings method
CN104542296B (en) Open rooting method for sugarcane tissue culture seedlings
CN1985578A (en) Tissue culture seedling producing process for lechang michelia and golden leaf michelia
CN115474552B (en) Tissue culture method for yellow She Monian hemp plants
CN102067818B (en) Inducing technology of test tube lotus root
CN101658133A (en) Method for quickly and directly establishing albizzia regeneration plant system
CN101836589B (en) Method of rapid propagation of populus

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C53 Correction of patent for invention or patent application
CB03 Change of inventor or designer information

Inventor after: Zhao Fengxia

Inventor after: Gao Xiangbin

Inventor after: Wang Zhengping

Inventor after: Li Yaoyu

Inventor after: Song Pengfei

Inventor after: Li Haifeng

Inventor after: Song Xueli

Inventor after: Meng Zhiyong

Inventor before: Zhao Fengxia

Inventor before: Gao Xiangbin

Inventor before: Wang Zhengping

Inventor before: Li Haifeng

Inventor before: Song Xueli

COR Change of bibliographic data

Free format text: CORRECT: INVENTOR; FROM: ZHAO FENGXIA GAO XIANGBIN WANG ZHENGPING LI HAIFENG SONG XUELI TO: ZHAO FENGXIA GAO XIANGBIN WANG ZHENGPING LI YAOYU SONG PENGFEI LI HAIFENG SONG XUELI MENG ZHIYONG

C14 Grant of patent or utility model
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20141112

Termination date: 20160108